CN103074281A - Enterococcus faecalis FJL19 and application thereof - Google Patents
Enterococcus faecalis FJL19 and application thereof Download PDFInfo
- Publication number
- CN103074281A CN103074281A CN2013100204339A CN201310020433A CN103074281A CN 103074281 A CN103074281 A CN 103074281A CN 2013100204339 A CN2013100204339 A CN 2013100204339A CN 201310020433 A CN201310020433 A CN 201310020433A CN 103074281 A CN103074281 A CN 103074281A
- Authority
- CN
- China
- Prior art keywords
- enterococcus faecalis
- fjl19
- acid bacteria
- group
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to an enterococcus faecalis FJL19 with the preservation number of CGMCC No. 6995. The invention further provides the enterococcus faecalis FJL19 with the effects of suppressing growth of escherichia coli in chicken intestinal canals and promoting the growths of chickens. According to the invention, the enterococcus faecalis FJL19 is separated from jejuna of cage rearing adult laying hens; the bacterial strain vigorously grows; 5.1 * 109 cfu/ml bacteria can be cultured in 18 hours and have antibacterial effects; antimicrobial proteins can be produced by using a culture medium, so that the number of the escherichia coli in the chicken intestinal canals is lowered; a bacterium preparation is prepared through lactic acid bacteria to feed the chickens, so that the growths of the chickens can be improved; therefore, the enterococcus faecalis FJL19 provided by the invention has the advantages of important social significance and economic values in animal feeding, in particular development of novel poultry feed additive and researches on antibiotic alternatives.
Description
Technical field
The invention belongs to the animal microorganism field, relate to strain enterococcus faecalis FJL19 and an application thereof, particularly strain enterococcus faecalis FJL19 and the application thereof of from the chicken enteron aisle, separating.
Background technology
Milk-acid bacteria is the important probiotic bacterium of a class, is the important dominant microflora of a class in the animal intestinal.Lactic acid bacteria formulation is as a kind of novel green animal probiotics, nontoxic because of it, without resistance, noresidue, having no side effect enjoys the extensive concern of feed circle.
Adopt milk-acid bacteria as the probiotic bacterium feeding animals, except because milk-acid bacteria has certain trophism and the adhesive attraction to enteron aisle, be that mainly milk-acid bacteria is inhibited for some spoilage organism and harmful bacteria.The first, milk-acid bacteria can produce lactic acid, creates sour environment and suppresses harmful bacteria and the growth of acid nonfast spoilage organism; The second, milk-acid bacteria produces H
2O
2, activate catalase-thiocyanic acid system, suppress and kill Gram-negative bacteria, catalase positive bacteria etc.; The 3rd, the part milk-acid bacteria can produce tiny protein or the peptide class of biocidal property, is called bacteriocin, and the pathogenic bacterium such as intestinal bacteria are had antagonistic action.And, the milk-acid bacteria that produces antibacterial albumen has been arranged, just can produce bacteriocin, can substitute antibiotics, it is safer that animal is produced.The milk-acid bacteria that separation screening can produce bacteriocin becomes the study hotspot that present probiotic bacterium is developed.
The originating in lactic acid bacterium that produces bacteriocin is a lot, and the industrial strain of purchase and reference culture have much also can produce bacteriocin, can the separating lactic acid bacterium in environment, soil, pickles, the sour milk.Yet probiotic bacterium is wanted feeding animals at last, and with respect to the bacterial strain in other sources, animal is undoubtedly the preferably source of milk-acid bacteria so.Milk-acid bacteria from animal intestinal can adapt to the animal intestinal environment, and the hydrochloric acid in gastric juice of tolerance animal and the digestion of cholate are undoubtedly best probiotic bacterium source.As seen, will be the important research direction that animal is screened with probiotic bacterium by the milk-acid bacteria that can produce bacteriocin from the animal intestinal Isolation and screening.
Summary of the invention
The purpose of this invention is to provide a strain enterococcus faecalis (Enterococcus faecalis) FJL19, its deposit number is CGMCC No.6995.
Second purpose of the present invention provides above-mentioned enterococcus faecalis FJL19 and has the effect that suppresses Escherichia coli Growth in the chicken enteron aisle, promotes chick growth.
The present invention is achieved through the following technical solutions:
One, a strain enterococcus faecalis (Enterococcus faecalis) FJL19, its deposit number is CGMCC No.6995.
Enterococcus faecalis FJL19 is Gram-positive among the present invention, and form is spherical, is the lactic bacterium strains that separates from the adult laying hen cecal content of raising in cages, the aimed strain that obtains through producing the characteristic measurement screenings such as antibacterial albumen.
The concrete steps of screening are as follows:
1, milk-acid bacteria separation and purification:
Adopt aseptic technique, with the slide glass scraping laying hen cecal content 1g that grows up that raises in cages, place the glass test tube that fills the 9mL stroke-physiological saline solution, fully concussion shakes up, then draw the 0.5mL mixed solution in the test tube that fills the 4.5mL stroke-physiological saline solution, this extent of dilution is 10
-1, repeat above process and make 10 doubling dilutions, to 10
-6Weaker concn selects 10
-4, 10
-5, 10
-6Three extent of dilution are drawn 0.lmL bacterium drop on the MRS culture medium flat plate, adopt methods,anaerobic (5%CO after the dull and stereotyped coating
2) culture dish that coats is put 37 ℃ of incubators cultivation 48h.Adopt four sectional streak methods, with the different bacterium colony of transfering loop picking form in the MRS nutrient agar separation and Culture of ruling, after 48h cultivates, the bacterium colony of picking good separating effect is inoculated in the MRS slant medium with transfering loop and does pure culture in four subregions, pure culture 3 times that repeat to go down to posterity, and it is for subsequent use to be placed on 4 ℃ of Refrigerator stores.
2, the observation of thalli morphology
Get clean slide, get a ring sterilized water on slide with transfering loop, then a small amount of bacterium of picking smoothens, to be dried after, fixing.Gramstaining: with ammonium oxalate crystal violet dye liquor dyeing 1min, the water flushing then drips Lushi's iodine liquid and covers 1min first, the water flushing, then drip 95% ethanol and when ethanol does not present purple, do not stop about 0.5min, redye 1min with the sarranine dye liquor at last, washing.Oil mirror microscopy (16 * 100) chooses that gramstaining is positive, form is that shaft-like or spherical bacterial strain is done the reserve bacterial strain and continued to employ.
The preparation of gramstaining reagent:
1. the preparation of ammonium oxalate crystal violet staining fluid
A liquid Viola crystallina 2g 95% alcohol 20mL
B liquid ammonium oxalate 0.8g distilled water 80mL
Mix A, B liquid, leave standstill 48h and use afterwards.
2. Lushi's iodine liquid
Crystalline flake of iodine 1g potassiumiodide 2g distilled water 300mL
Potassiumiodide is dissolved in a small amount of water first, again the crystalline flake of iodine is dissolved in the liquor kalii iodide, after iodine is entirely molten, fill up water and get final product.
3. 95% alcohol
4. sarranine is redyed liquid
Sarranine 2.5g 95% alcohol 100mL
Getting the above-mentioned sarranine alcohol 10mL for preparing and 80mL distilled water mixing forms.
3, biocidal property lactic acid screening:
(1) preparation of bacterium liquid: the milk-acid bacteria activation 2-3 that separation and purification is gone out is after generation, is connected to separately in the fresh MRS liquid nutrient medium by the inoculum size of 2% (v/v), and 37 ℃ of anaerobism cultivation 24h make its bacterial concentration reach 10
8Cfu/mL measures its bacteriostatic activity.Micrococcus flavus is inoculated in the broth culture, cultivated for 2 generations in 37 ℃ of shaking tables, make 10 doubling dilutions with sterilized water, make its bacterial concentration reach 10
5Cfu/mL, for subsequent use as indicator.
(2) acid producing ability is measured: the milk-acid bacteria that separation and purification is gone out activates 2-3 after generation, be connected to separately in the fresh MRS liquid nutrient medium by the inoculum size of 2% (v/v), 37 ℃ of anaerobism are cultivated, and measure the pH value of each experimental strain 5mL fermented liquid with acidometer respectively at two time points of 0h, 48h.Each test strain fermented liquid is done 3 repetitions, takes the mean.PH value with two each experimental strain fermented liquids of time point of 0h, 48h changes, and namely △ pH value is weighed the acid producing ability of milk-acid bacteria.Choose the high bacterial strain of acid producing ability and carry out next step mensuration.
(3) bacteriostatic experiment: the milk-acid bacteria of adopting the Oxford agar diffusion method that separation and purification is gone out carries out fungistatic effect and measures.
Get 100 μ L indicator liquid and be added drop-wise to respectively and solidified in the good substratum, with the spreading rod coating evenly.Be placed on the plate with 4 Oxfords of aseptic nipper gripping cup symmetry under aseptic condition, then draw respectively 200 μ L streptococcus acidi lactici fermented solutions in 3 Oxford cups, the MRS nutrient solution that adds equivalent in another Oxford cup compares, in 37 ℃ of lower constant temperature culture 16-18h.Each streptococcus acidi lactici fermented solution is done 2 repetitions, surveys the size of its antibacterial circle diameter with vernier callipers, takes the mean, and selects the large milk-acid bacteria of antibacterial ring.
In order to filter out as early as possible required bacterial classification, preliminary screening has adopted antibacterial and 2 basic indexs of acid producing ability add and concentration.In every index, what rank the first adds 100 minutes, occupies second add 99 minutes, by that analogy, comes last add 1 minute.2 results of Primary Screening Test are summed up separately and sort, select the higher top 10 bacterial strain of total points and carry out next step research.
4, the screening of bacteriocin-producing lactic acid bacteria
The milk-acid bacteria that screens in the step 2 is carried out liquid culture, in order to reduce the fungistatic effect of acid, nutrient solution is adjusted pH value to 6.8 with 1mol/L NaOH solution, then carry out the Oxford agar diffusion method and measure antibacterial ring, method is the same, selects the maximum milk-acid bacteria of antibacterial ring.After carrying out liquid culture again, add 7 ℃ of effects of protease 3 2h of 0.1mg/L in nutrient solution, get bacterium liquid and do bacteriostatic test, screening does not have the bacterial strain of antibacterial ring, for producing the target milk-acid bacteria of antibacterial albumen.
In above-mentioned screening method, substratum is the MRS substratum:
Solid plate or slant medium: Tryptones 10g, extractum carnis 10g, yeast extract paste 5g, ammonium citrate 2g, glucose 20g, potassium primary phosphate 2g, sodium acetate 5g, tween-80 1g, sal epsom 0.5g, manganous sulfate 0.2g, agar 20g, distilled water 1000mL, pH6.8.
Liquid nutrient medium: Tryptones 10g, extractum carnis 10g, yeast extract paste 5g, ammonium citrate 2g, glucose 20g, potassium primary phosphate 2g, sodium acetate 5g, tween-80 1g, sal epsom 0.5g, manganous sulfate 0.2g, distilled water 1000mL, pH6.8.
Adopt the positively effect of technique scheme: the present invention isolates enterococcus faecalis FJL19 from the adult laying hen jejunum of raising in cages, this strain growth is vigorous, and breeding bacteria just can reach 5.1 * 10 in 18 hours
9Cfu/mL, and have bacteriostatic action, can utilize substratum to produce antibacterial albumen, reduce chick enteron aisle intestinal bacteria quantity; Prepare the bacteria preparation feeding chickling by this milk-acid bacteria, can improve the growth of chick; Therefore enterococcus faecalis FJL19 has great social effect and economic worth in animal rearing especially poultry novel fodder additive exploitation and Substitutes For Antibiotic research.
Description of drawings
Fig. 1 is the gram stain microscopy result of enterococcus faecalis;
Fig. 2 is the fungistatic effect contrast of enterococcus faecalis bacterium liquid;
Among the figure, 1,2 is the inhibition zone of purpose bacterial strain, and 3,4 is the inhibition zone of other isolated strains;
Fig. 3 is the fungistatic effect of enterococcus faecalis bacterium liquid after the protease treatment;
Among the figure, 1 is untreated bacterium liquid, and 2,3 and 4 are respectively Proteinase K, the bacterium liquid that Trypsin and papoid are processed;
Fig. 4 is the pcr amplification result of enterococcus faecalis;
Among the figure, 1Marker, 2 plant lactobacilluss;
Fig. 5 is the growth curve chart of enterococcus faecalis.
Enterococcus faecalis involved in the present invention (Enterococcus faecalis) FJL19, carried out the patented procedure preservation on December 17th, 2012 at the preservation center that Patent Office of the People's Republic of China or international monopoly tissue are admitted, depositary institution's full name is China Committee for Culture Collection of Microorganisms common micro-organisms center, referred to as CGMCC, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number: CGMCC No.6995.
Embodiment
Below in conjunction with embodiment, test example technical scheme of the present invention is described further, but should not be construed as limitation of the present invention:
The source of biomaterial among the present invention:
1, the bacterium universal primer is available from Shanghai Bo Ya Bioisystech Co., Ltd.
Present embodiment is used for the screening of explanation enterococcus faecalis FJL19.
1, milk-acid bacteria separation and purification:
Adopt aseptic technique, with the slide glass scraping laying hen cecal content 1g that grows up that raises in cages, place the glass test tube that fills the 9mL stroke-physiological saline solution, fully concussion shakes up, then draw the 0.5mL mixed solution in the test tube that fills the 4.5mL stroke-physiological saline solution, this extent of dilution is 10
-1, repeat above process and make 10 doubling dilutions, to 10
-6Weaker concn selects 10
-4, 10
-5, 10
-6Three extent of dilution are drawn 0.lmL bacterium drop on the MRS culture medium flat plate, adopt methods,anaerobic (5%CO after the dull and stereotyped coating
2) culture dish that coats is put 37 ℃ of incubators cultivation 48h.Adopt four sectional streak methods, with the different bacterium colony of transfering loop picking form in the MRS nutrient agar separation and Culture of ruling, after 48h cultivates, the bacterium colony of picking good separating effect is inoculated in the MRS slant medium with transfering loop and does pure culture in four subregions, pure culture 3 times that repeat to go down to posterity, and it is for subsequent use to be placed on 4 ℃ of Refrigerator stores.
2, the observation of thalli morphology
Get clean slide, get a ring sterilized water on slide with transfering loop, then a small amount of bacterium of picking smoothens, to be dried after, fixing.Gramstaining: with ammonium oxalate crystal violet dye liquor dyeing 1min, the water flushing then drips Lushi's iodine liquid and covers 1min first, the water flushing, then drip 95% ethanol and when ethanol does not present purple, do not stop about 0.5min, redye 1min with the sarranine dye liquor at last, washing.Oil mirror microscopy (16 * 100) chooses that gramstaining is positive, form is that shaft-like or spherical bacterial strain is done the reserve bacterial strain and continued to employ.The result as shown in Figure 1.
The preparation of gramstaining reagent:
1. the preparation of ammonium oxalate crystal violet staining fluid
A liquid Viola crystallina 2g 95% alcohol 20mL
B liquid ammonium oxalate 0.8g distilled water 80mL
Mix A, B liquid, leave standstill 48h and use afterwards.
2. Lushi's iodine liquid
Crystalline flake of iodine 1g potassiumiodide 2g distilled water 300mL
Potassiumiodide is dissolved in a small amount of water first, again the crystalline flake of iodine is dissolved in the liquor kalii iodide, after iodine is entirely molten, fill up water and get final product.
3. 95% alcohol
4. sarranine is redyed liquid
Sarranine 2.5g 95% alcohol 100mL
Getting the above-mentioned sarranine alcohol 10mL for preparing and 80mL distilled water mixing forms.
3, biocidal property lactic acid screening:
(1) preparation of bacterium liquid: the milk-acid bacteria activation 2-3 that separation and purification is gone out is after generation, is connected to separately in the fresh MRS liquid nutrient medium by the inoculum size of 2% (v/v), and 37 ℃ of anaerobism cultivation 24h make its bacterial concentration reach 10
8Cfu/mL measures its bacteriostatic activity.Micrococcus flavus is inoculated in the broth culture, cultivated for 2 generations in 37 ℃ of shaking tables, make 10 doubling dilutions with sterilized water, make its bacterial concentration reach 10
5Cfu/mL, for subsequent use as indicator.
(2) acid producing ability is measured: the milk-acid bacteria that separation and purification is gone out activates 2-3 after generation, be connected to separately in the fresh MRS liquid nutrient medium by the inoculum size of 2% (v/v), 37 ℃ of anaerobism are cultivated, and measure the pH value of each experimental strain 5mL fermented liquid with acidometer respectively at two time points of 0h, 48h.Each test strain fermented liquid is done 3 repetitions, takes the mean.PH value with two each experimental strain fermented liquids of time point of 0h, 48h changes, and namely △ pH value is weighed the acid producing ability of milk-acid bacteria.Choose the high bacterial strain of acid producing ability and carry out next step mensuration.
(3) bacteriostatic experiment: the milk-acid bacteria of adopting the Oxford agar diffusion method that separation and purification is gone out carries out fungistatic effect and measures.
Get 100 μ L indicator liquid and be added drop-wise to respectively and solidified in the good substratum, with the spreading rod coating evenly.Be placed on the plate with 4 Oxfords of aseptic nipper gripping cup symmetry under aseptic condition, then draw respectively 200 μ L streptococcus acidi lactici fermented solutions in 3 Oxford cups, the MRS nutrient solution that adds equivalent in another Oxford cup compares, in 37 ℃ of lower constant temperature culture 16-18h.Each streptococcus acidi lactici fermented solution is done 2 repetitions, surveys the size of its antibacterial circle diameter with vernier callipers, takes the mean, and selects the large milk-acid bacteria of antibacterial ring.
In order to filter out as early as possible required bacterial classification, preliminary screening has adopted antibacterial and 2 basic indexs of acid producing ability add and concentration.In every index, what rank the first adds 100 minutes, occupies second add 99 minutes, by that analogy, comes last add 1 minute.2 results of Primary Screening Test are summed up separately and sort, select the higher top 10 bacterial strain of total points and carry out next step research.
4, the screening of bacteriocin-producing lactic acid bacteria
The milk-acid bacteria that screens in the step 2 is carried out liquid culture, in order to reduce the fungistatic effect of acid, nutrient solution is adjusted pH value to 6.8 with 1mol/L NaOH solution, then carry out the Oxford agar diffusion method and measure antibacterial ring, method is the same, selects the maximum milk-acid bacteria of antibacterial ring.The result as shown in Figure 2.After carrying out liquid culture again, add 7 ℃ of effects of protease 3 2h of 0.1mg/L in nutrient solution, get bacterium liquid and do bacteriostatic test, screening does not have the bacterial strain of antibacterial ring, for producing the target milk-acid bacteria of antibacterial albumen.The result as shown in Figure 3.
In above-mentioned screening method, substratum is the MRS substratum:
Solid plate or slant medium: Tryptones 10g, extractum carnis 10g, yeast extract paste 5g, ammonium citrate 2g, glucose 20g, potassium primary phosphate 2g, sodium acetate 5g, tween-80 1g, sal epsom 0.5g, manganous sulfate 0.2g, agar 20g, distilled water 1000mL, pH6.8.
Liquid nutrient medium: Tryptones 10g, extractum carnis 10g, yeast extract paste 5g, ammonium citrate 2g, glucose 20g, potassium primary phosphate 2g, sodium acetate 5g, tween-80 1g, sal epsom 0.5g, manganous sulfate 0.2g, distilled water 1000mL, pH6.8.
Present embodiment is used for the evaluation of explanation enterococcus faecalis FJL19.
1, bacterial strain Identification
Catalase test: with 3%H
2O
2Directly be added on the lawn on inclined-plane, observe whether alveolate generation.If then positive reaction of Bubble formation is arranged.
Nitrate reduction test: microbionation to substratum, is cultivated 48h for 37 ℃, add first liquid and second liquid along tube wall, observe.Present at once redness, orange red positive reaction.
Gelatin liquification test: microbionation to substratum, is cultivated 48h for 20 ℃, observe.The inoculated tube positive reaction of liquefying.
Indole test: inoculated bacteria is cultivated 48h for 37 ℃ in the MRS nutrient solution.Add dimethylbenzene 2-3mL in nutrient solution, shake up, leave standstill moments later, add indoles reagent 2mL along tube wall, dimethylbenzene lower floor liquid becomes the positive reaction of rose person.
Hydrogen sulfide production test: microbionation behind the MRS nutrient solution, is hung in the inoculated tube with aseptic tweezers gripping one lead acetate paper slip.The lower end does not contact liquid level near media surface.The upper end is filled in tampon.Place 37 ℃ to cultivate 48h, observe.The positive reaction of paper slip blackening.
By catalase test, nitrate reduction test, gelatin liquification test, indole test and hydrogen sulfide production test, the result shows all negative, illustrates that enterococcus faecalis FJL19 is genus lactubacillus.
2, the evaluation of bacterial strain kind
Sugar fermentation is produced sour aerogenesis test: in lactose, D-wood sugar, sucrose, melibiose, raffinose, melizitose, ribitol, sorbyl alcohol and N.F,USP MANNITOL biochemical fermentation pipe, cultivate 48h with a small amount of microbionation of inoculating needle difference picking for 37 ℃.The purple yellowing represents to produce acid, positive reaction in the biochemical tube.
15 ℃ and 45 ℃ of growth tests: whether a few ring microbionations of picking are cultivated 48h for 15 ℃ and 45 ℃ on the MRS slant medium, observe bacterium and grow on the inclined-plane.
Arginine hydrolysis experiment: microbionation to substratum, is cultivated 48h for 37 ℃, observe, present redness, orange red positive reaction.
Hippurate hydrolysis experiment: microbionation to substratum, is cultivated 48h for 37 ℃, observe.Present red, orange red positive reaction.
Arginine produces ammonia test: inoculated bacteria is cultivated 48h for 37 ℃ in containing arginic substratum.Add Nessler's reagent number droplet in the nutrient solution, when producing ammonia, occur orange or the tawny precipitation, be positive reaction.
By hippurate hydrolysis experiment, arginine hydrolysis experiment, 15 ℃ and 45 ℃ of growth tests, and all kinds of sugar fermentating tests, result and " common bacteria system identification handbook and " classification of lactic-acid-bacterium is identified " contrast.Qualification result shows that this bacterial strain is enterococcus faecalis.
In above-mentioned screening method, biochemical medium and related reagent are formulated as follows:
Hydrogen sulfide production test substratum: tryptone 10g; Extractum carnis 3g; Yeast extract 5g; NaCl 5g; Halfcystine 0.4g; Glucose 2g; Distilled water 1000mL.Adjust pH is in 7.2,115 ℃ of autoclaving 20min.
Lactose, D-wood sugar, rhamnosyl, sucrose, melibiose, raffinose, melizitose, ribitol, sorbyl alcohol and N.F,USP MANNITOL sugar-fermenting are identified biochemical tube, are rich biological purchase the in sea.
Indoles reagent: Paradimethylaminobenzaldehyde 1g, dehydrated alcohol 95mL, concentrated hydrochloric acid 20mL.
Arginine liquid: L-arginine 1.5g, halfcystine (1g/mL H
2O) 0.05mL, distilled water 10mL transfers pH to 7.0, adds 3 after the sterilization and drops in the PY basic culture solution.Nessler's reagent: 20g KI is dissolved in 50mL distilled water, and in this solution, adds HgI
2Small-particle, to solution reach saturated till (about 32g), and then add 460mL water and 134gKOH.Be stored in the shaded bottle supernatant liquor for subsequent use.
Experimental result such as the table 1 of sugar-fermenting:
Table 1 sugar fermentating test result
The evaluation 3.16S rDNA checks order
Employing well known to a person skilled in the art method, extracts bacterial genomes DNA with test kit, and reverse transcription is cDNA, and the conservative region according to the 16S rDNA gene order of lactobacillus adopts the bacterium universal primer, carries out the conventional pcr amplification of gene, wherein,
Upstream primer 5'-AGAGTTTGATCCTGGCTCAG-3'
Downstream primer 5'-ACGGCTACCTTGTTACGACTT3'
The amplification electrophoresis detection, the electrophoresis showed band is clear single, illustrates and increases successfully, result such as Fig. 4, band is purified simultaneously, the TA-clone technology, after the order-checking, carry out sequence alignment at the BLAST of Gene bank, be defined as enterococcus faecalis (Enterococcus faecalis).
Test example 1
This test example is used for the effect of explanation enterococcus faecalis FJL19.
1, the full bacterium liquid formulation preparation of milk-acid bacteria
(1) lactobacillus-fermented timing: the Bacterium lacticum after will activating is as fermentation seed liquid 1%(v/v) be inoculated in the MRS liquid nutrient medium, 37 ℃, the 170r/min shaking table is cultivated, at front 24h every 2h, behind the 24h the 36th and 48h sampling, after carrying out gradient dilution by 10 times of methods with 0.9% physiological saline, get 10
-4-10
-6Extent of dilution 100 μ l diluents evenly are coated with at the MRS nutrient agar, and each gradient is done 3 repetitions, and plate is at 37 ℃, 5%CO
2Cultivate 24h under the condition.Get colony number and calculate bacterial concentration in the extent of dilution value of 30-300, simultaneously sampling adopts the Oxford agar diffusion method to measure the fungistatic effect of each time point bacterium liquid, determines the suitableeest fermentation time.
As shown in Figure 5, X-coordinate represents the time among the figure, and ordinate zou represents viable count, and enterococcus faecalis viable count rising in the 10h after inoculation then presents the state of rapid rising, and reached viable count 10 when 18h behind 10h
9More than, reached maximum this moment.And viable count has dropped to 10 when having arrived 36h
8, and present the state that descends gradually.Major cause is the consumption of substratum nutritive substance, and the reasons such as harmful Metabolite Accumulation increase cause the thalli growth environmental evolution, thereby number of viable is reduced.Can find out that from growth curve enterococcus faecalis viable count when 16-20h maintains 10
9The order of magnitude, and more stable, so select this time period results thalline of 18h.
(2) inoculum size full bacterium solution preparation: test strain is activated 2-3 after generation, by 1%(v/v) is connected in the liquid nutrient medium of optimization 37 ℃, the 170r/min shaking table is cultivated 18h, 4 ℃, the centrifugal 30min of 3500rpm, with the supernatant liquor suspension thalline of certain volume, 4 ℃ save backup.
2, lactic acid bacteria formulation is at the effect of breeding chickling
(1) test materials: above-mentioned full bacterium liquid formulation, viable count is 5.1 * 10
9Cfu/mL(bacterium number); Microbiotic is 4% bambermycin.
(2) experimental animal and test design: select the AA broiler chicken of 240 1 age in days health, male and female half and half, mean body weight 40.55 ± 0.67g, be divided at random 4 groups, I group (control group, basal diet), II group (microbiotic group, basal diet+0.01% bambermycin), III (full bacterium liquid formulation group, the full bacterium liquid formulation of basal diet+0.5%), IV group (full bacterium liquid formulation group, the full bacterium liquid formulation of basal diet+1%), every group of 5 repetitions, each repeats 12 chickens, 28 days trial periods.
(3) testing index:
A, production performance index: each repetition of weighing on an empty stomach heavy (fasting 12h) during respectively at test 8:00 the 1st day, 15 days, 29 days morning, and accurate recording respectively repeats food consumption.Calculate the average daily gain (ADG) of respectively organizing chicken, average daily ingestion amount (ADFI) and feed conversion ratio (FCR).Result such as table 2:
Table 2 lactic acid bacteria formulation is on the impact of chick day weight gain and material anharmonic ratio
Notes: the colleague compares, different lowercase alphabet differentials different significantly (p＜0.05), and different capitalizations represent difference extremely significantly (p＜0.01), unmarked or alphabetical identical person represents difference not remarkable (p＞0.05).
Test-results shows, 1～14d and 15～28d, 1.0% lactic acid bacteria formulation group broiler chicken average daily gain is significantly higher than control group (p＜0.05), the a little higher than control group of 0.5% lactic acid bacteria formulation group average daily gain, but difference is (p〉0.05) not significantly, average daily gain difference not significantly (p〉0.05) between the preparation group, 1～14d, average daily gain is without significant difference (p〉0.05) between each processing of microbiotic group and other; 15～28d, the average daily gain of microbiotic group is significantly higher than control group (p＜0.05), but processes group difference all not significantly (p〉0.05) with other.Test the full phase, the average daily gain of lactic acid bacteria formulation group all is significantly higher than control group (p＜0.05), 1.0% preparation group is significantly higher than 0.5% preparation group (p＜0.05), the average daily gain of microbiotic group is significantly higher than control group and 0.5% lactic acid bacteria formulation group (p＜0.05), with 1% lactic acid bacteria formulation group difference not significantly (p〉0.05).
15～28d, 1.0% lactic acid bacteria formulation group feedstuff-meat ratio all significantly is lower than control group (p＜0.05), and between microbiotic group and 0.5% preparation group without significant difference (p〉0.05), feedstuff-meat ratio difference all not significantly (p〉0.05) between 0.5% preparation group, microbiotic and the control group.Test the full phase, preparation group feedstuff-meat ratio all significantly is lower than control group (p＜0.05), 1.0% preparation group feedstuff-meat ratio significantly is lower than 0.5% preparation group (p＜0.05), and microbiotic group feedstuff-meat ratio significantly is lower than control group (p＜0.05), with other treatment group difference not significantly (p〉0.05).
In general, lactic acid bacteria formulation and microbiotic all can improve the growth performance of broiler chicken, and 1.0% preparation group is better than 0.5% preparation group and microbiotic group.
B, caecum Microflora: in test the 15th, 29 day, respectively repeating to get one near the examination chicken of mean body weight at random, the jugular vein sacrificed by exsanguination, dissect immediately, with taking out behind the sterile cotton toe-in bundle caecum, take by weighing cecal content 0.5g under the aseptic condition, 10 times of dilutions, spread plate is measured milk-acid bacteria and intestinal bacteria quantity in the caecum.Result such as table 3:
Table 3 lactic acid bacteria formulation is on the impact (log of milk-acid bacteria in the chick caecum and intestinal bacteria quantity
10Cfu/g)
Notes: the colleague compares, different lowercase alphabet differentials different significantly (p＜0.05), and different capitalizations represent difference extremely significantly (p＜0.01), unmarked or alphabetical identical person represents difference not remarkable (p＞0.05).
During 2w, milk-acid bacteria bacterium digital display work is higher than control group and microbiotic group (p＜0.05) in the 1.0% lactic acid bacteria formulation group broiler chicken caecum, but compares with its 0.5% lactic acid bacteria formulation group, and difference is (p〉0.05) not significantly; 0.5% lactic acid bacteria formulation group milk-acid bacteria bacterium digital display work is higher than microbiotic group (p＜0.05), but compares with control group without significant difference (p〉0.05); The control group lactic acid bacteria number is significantly higher than microbiotic group (p＜0.05).1.0% lactic acid bacteria formulation group coliform count significantly is lower than other all treatment group (p＜0.05); 0.5% preparation group coliform count is compared difference not significantly (p〉0.05) with control group, microbiotic group coliform count significantly is lower than control group (p＜0.05), coliform count difference not significantly (p〉0.05) between 0.5% preparation group and microbiotic group.
During 4w, 1% and 0.5% lactic acid bacteria formulation group lactic acid bacteria number is significantly higher than microbiotic group (p＜0.05), and except the microbiotic group, lactic acid bacteria number is without significant difference (p〉0.05) between all the other each group; 0.5% and 1.0% lactic acid bacteria formulation group coliform count significantly is lower than other treatment group (p＜0.05).Along with the increase of lactic acid bacteria formulation addition, lactic acid bacteria number increases in the caecum, and coliform count reduces, and this shows that milk-acid bacteria can be optimized intestinal microflora.
To sum up, this bacterial strain can significantly suppress the intestinal bacteria quantity in the chicken enteron aisle, promotes simultaneously chick growth, and alternative traditional microbiotic reduces the application of microbiotic in animal rearing, has greatly improved biological safety.
Claims (2)
1. a strain enterococcus faecalis (Enterococcus faecalis) FJL19, its deposit number is CGMCC No.6995.
2. enterococcus faecalis FJL19 claimed in claim 1 has the effect that suppresses Escherichia coli Growth in the chicken enteron aisle, promotes chick growth.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100204339A CN103074281A (en) | 2013-01-21 | 2013-01-21 | Enterococcus faecalis FJL19 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100204339A CN103074281A (en) | 2013-01-21 | 2013-01-21 | Enterococcus faecalis FJL19 and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103074281A true CN103074281A (en) | 2013-05-01 |
Family
ID=48150980
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013100204339A Pending CN103074281A (en) | 2013-01-21 | 2013-01-21 | Enterococcus faecalis FJL19 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103074281A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774782A (en) * | 2014-12-25 | 2015-07-15 | 东北农业大学 | Chicken-sourced enterococcus faecalis separating strain and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1771831A (en) * | 2004-11-12 | 2006-05-17 | 浙江大舜恒益生物技术有限公司 | Feed-grade microbial additive and preparation and application thereof |
| CN101220342A (en) * | 2007-11-14 | 2008-07-16 | 大连三仪动物药品有限公司 | FQ15 enterococcus faecalis and method for producing somatotrophic feed additive with the bacteria |
| CN101810247A (en) * | 2009-12-30 | 2010-08-25 | 沈阳科丰牧业科技有限公司 | Complex microbial chicken feed additive |
| CN102031235A (en) * | 2010-11-09 | 2011-04-27 | 中国农业大学 | Enterococcus faecium ANSE228 and application thereof |
| CN102657288A (en) * | 2012-03-12 | 2012-09-12 | 北京大北农科技集团股份有限公司 | Formula feed for chickens and preparation method thereof |
| CN102732468A (en) * | 2012-07-06 | 2012-10-17 | 北京农学院 | Enterococcus faecalis and broad-spectrum antibacterial action thereof |
-
2013
- 2013-01-21 CN CN2013100204339A patent/CN103074281A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1771831A (en) * | 2004-11-12 | 2006-05-17 | 浙江大舜恒益生物技术有限公司 | Feed-grade microbial additive and preparation and application thereof |
| CN101220342A (en) * | 2007-11-14 | 2008-07-16 | 大连三仪动物药品有限公司 | FQ15 enterococcus faecalis and method for producing somatotrophic feed additive with the bacteria |
| CN101810247A (en) * | 2009-12-30 | 2010-08-25 | 沈阳科丰牧业科技有限公司 | Complex microbial chicken feed additive |
| CN102031235A (en) * | 2010-11-09 | 2011-04-27 | 中国农业大学 | Enterococcus faecium ANSE228 and application thereof |
| CN102657288A (en) * | 2012-03-12 | 2012-09-12 | 北京大北农科技集团股份有限公司 | Formula feed for chickens and preparation method thereof |
| CN102732468A (en) * | 2012-07-06 | 2012-10-17 | 北京农学院 | Enterococcus faecalis and broad-spectrum antibacterial action thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774782A (en) * | 2014-12-25 | 2015-07-15 | 东北农业大学 | Chicken-sourced enterococcus faecalis separating strain and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104293696B (en) | One strain enterococcus faecalis HEW-A131 and application thereof | |
| CN103614327B (en) | A kind of subtilis and uses thereof | |
| CN103981118B (en) | A kind of bacillus subtilis feed addictive and its preparation method and application | |
| CN101690545B (en) | Method for producing complex micro-ecological preparation with microbial agents and enzyme | |
| CN109749957B (en) | Preparation and application of lactobacillus gasseri preparation with aquatic pathogenic bacteria antagonistic property | |
| CN109161509B (en) | Bacterial strain capable of preventing and treating bovine and sheep diarrhea | |
| CN109337841A (en) | One plant of bacillus subtilis BYS2 with Efficient antibacterial performance | |
| CN103013893A (en) | Lactobacillus plantarum CCL67 and application of same | |
| CN107964524B (en) | Lactococcus lactis HKS2 with lactic acid activity and separation and screening method and application thereof | |
| CN101392223A (en) | Breeding method of microbial feed additive strain | |
| CN105524855B (en) | A kind of bacillus coagulans and its application with aquatic pathogenic bacterium antagonistic properties | |
| CN106119152A (en) | The bacillus acidophilus of a kind of high-yield lactic acid rhzomorph and application thereof | |
| CN101884394B (en) | Composite microbial feed additive for chicken and preparation method thereof | |
| WO2019205422A1 (en) | Bacillus coagulans xp and application thereof in animal feed production | |
| CN102517227A (en) | Enterococcus faecalis and applications and feed additive and leavening agent thereof | |
| CN102329749A (en) | Bacillus subtilis bred by space mutation breeding technology and application of Bacillus subtilis | |
| CN114085789B (en) | Pediococcus pentosaceus MA.WTPQJ01 and application thereof | |
| CN103266074B (en) | B.subtilis spores strain and application thereof | |
| CN103082156A (en) | Ruminant compound microecological preparation and application thereof | |
| CN105199978A (en) | Bacillus coagulans preparation for livestock breeding and preparation method thereof | |
| CN110016443B (en) | Lactobacillus reuteri and application thereof in production of selenium-rich eggs | |
| CN103146602A (en) | Lactic acid bacteria solid co-fermenting method, obtained bacterial agent and use of bacterial agent | |
| CN106509468A (en) | Composite type synbiotic microecological preparation for chickens and preparation method of composite type synbiotic microecological preparation | |
| CN114231464B (en) | Bacillus coagulans and application thereof | |
| CN102669432A (en) | Compound probiotics preparation for geese and method for preparing same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130501 |